Background
Zika virus (ZIKV) belongs to the
Flavivirus genus and is related to other viruses that are also transmitted by the bite of mosquitoes, such as dengue virus (DENV), yellow fever virus (YFV) and West Nile virus (WNV). The
Flaviviridae family comprises single-strand RNA, membrane-enveloped viruses that frequently use
Aedes aegypti as a vector. Despite ZIKV being discovered over 60 years ago, only since 2014 (in the French Polynesia Islands) and 2015 (Brazil and America) has it been evident that the virus can cause large outbreaks and epidemics that lead to a global public health emergency [1].
ZIKV infection causes a mild severity, undifferentiated febrile syndrome, characterized by rash, arthralgia, myalgia and conjunctivitis, symptoms that are similar to those that appear in DENV fever or chikungunya virus (CHIKV) fever (CHIKV being an unrelated alphavirus transmitted by the same mosquito). The similarities of the symptoms causes confusion between the diseases during clinical evaluation. Also, these three viral illnesses may co-circulate in the same areas, hampering the final diagnosis of patients.
Although the ZIKV morbidity and mortality are considered low, it was demonstrated during the recent outbreaks that infection in pregnant women may be associated with severe birth defects (mostly microcephaly), and with the appearance in infected adults of a severe neurologic disease called Guillain–Barré syndrome (GBS). This neurologic entity increased 2–10-fold the historic cases in Latin America during the 2016 ZIKV epidemic [2]. Epidemiological estimates consider that approximately 75% of ZIKV-infected people do not present signs or symptoms during an outbreak, but they become an efficient transmission focus to mosquitoes and other individuals.
It is well known that mosquito bites are the main transmission route in areas where the insect infestation rates are high; however, it recently has been confirmed that ZIKV is capable of crossing the placental barrier and infecting the fetus. In adult patients, the virus persists in semen and vaginal fluids for two months, producing a viral load sufficient for transmission during sexual intercourse. This finding changes the epidemiological trends, as it is now also possible to detect infected patients in non-tropical countries, challenging the clinical and laboratory diagnosis. However, it is clear that tropical underdeveloped countries will still be the major source of febrile cases and, of course, the congenital malformations and GBS appearance in adults.
ZIKV infection diagnostics
The incubation period of ZIKV disease is not clear but is likely to be a few days, similar to other arboviruses. Symptoms can begin 2 to 7 days after a mosquito bite and last for 3 to 7 additional days. In both early symptomatic or asymptomatic cases, the virus can be detected by reverse transcription (RT)-PCR after purification of plasma or serum RNA. The acute sera can be inoculated in Vero cells or C6/36 mosquito cells to attempt virus isolation, but although this technique is powerful, it is expensive and lacks clinical value. We successfully isolated ZIKV and produced enough inoculum for cell biology and immunologic studies (Fig. 1). As a result of their sensitivity and specificity, ZIKV RNA detection by different nucleic acid tests is used on a routine basis to confirm acute ZIKV cases.
RT-PCR
The real-time RT-PCR protocol designed by the Centers for Disease Control (CDC, USA) during the 2007 Yap Island outbreak is the most used and evaluated, even after the confirmation that a very low viral load occurs during the acute phase and that viremia lasts only a few days in both symptomatic and asymptomatic individuals. This CDC protocol does not amplify RNA from other flaviviruses and alphaviruses [3].
The test was designed as a one-step RT-PCR with fluorogenic probes using serum as the sample and is also used on urine samples, where the virus can be detected until 15 days after symptoms start and when the serum sample has become negative. A comparison between different sample types demonstrated that saliva may be better than serum for confirming ZIKV infection [4]. A very sensitive and specific synthetic biology tool based on isothermal amplification and toehold switch RNA sensors has been reported and is currently under evaluation in field conditions in Colombia, Brazil and Equator [5].
Many other real-time PCR tests have recently been developed, but there are no reports regarding their clinical evaluation. One test with excellent analytical performance is becoming available (Altona Diagnostics), but it has not yet reported clinical assays in ZIKV circulating zones.
Frequently, conventional PCR has been used to follow epidemics and ZIKV circulation in mosquitoes [6], and this reported test was used to confirm the first cases in Brazil. Recently, we used modified primers to perform a double-round one-step RT-PCR to detect DENV, ZIKV and CHIKV in the serum of febrile patients, obtaining samples simultaneously positive for two or even three viruses [7]. This test also detects ZIKV RNA in paired samples of serum, breast milk and urine (Fig. 2).
Serology
The main challenge to serological ZIKV diagnosis is related to its structural proximity to other flaviviruses (DENV, YFV, and WNV) because antibodies against one of them can recognize the other viruses on ELISA platforms, frequently resulting in a false positive diagnostic. For this reason, RNA detection is preferred to confirm the infection during the first week after symptoms appear. However, serological tests are recommended to facilitate the diagnosis of pregnant women living in endemic zones or women or couples wanting pre-conception counselling because ZIKV IgM positivity confirms previous exposure to the virus; in those who are negative, it is recommended to perform periodic tests to prove the absence of virus contact.
Since the first serological diagnostics were performed in Africa, there have been difficulties confirming the infection by antibodies due to cross-reactivity in neutralization tests, hemagglutination inhibition and mouse neutralization used in the 1950s [8]. During the Yap Islands’ 2007 ZIKV outbreak, in addition to molecular confirmation, 14 ZIKV cases were investigated by serology, and it was confirmed that 8/14 individuals who had a previous flavivirus infection (secondary flavivirosis) were positive by DENV IgM ELISA. In addition, ZIKV-confirmed sera had high titres in the plaque reduction neutralization test (PRNT), mainly to DENV (12/14), YFV (3/14) and WNV (6/14) [3].
Currently, the CDC uses IgM antibody capture (MAC)-ELISA in its diagnostic algorithm, in which the ZIKV antigen is obtained from infected mice brains or recombinant proteins (Fig. 3a). This test is being used to confirm recent infections and to counsel women in endemic zones. This ELISA has not yet been tested in endemic zones where other flaviviruses circulate. Recently, an assay based on non-structural protein NS1 from ZIKV adsorbed to ELISA plates has been reported (Euroimmun AG), showing excellent performance to detect both IgM and IgG using samples from endemic zones and samples with confirmed contact with other flaviviruses (Fig. 3b) [9].
Pregnant women: the priority
Considering that ZIKV infection during the first two trimesters of pregnancy can be associated with neurological defects in the fetus, it is important to evaluate the infection risk in three different groups of women: (i) women of childbearing age living in areas with virus circulation; (ii) women travelling frequently to endemic zones; and (iii) women having sexual intercourse with individuals travelling frequently to ZIKV endemic zones (Table 1). Notably, only 25% of infected individuals present signs or symptoms of the disease, but those who are asymptomatic can transmit the virus to mosquitoes and through sexual contact, can develop GBS or can transmit the virus to the fetus during pregnancy. Normally, health authorities do not recommend confirming all cases by PCR or serology, but only those needed to facilitate the surveillance of ZIKV infections or sequelae (Table 1).
CDC testing algorithm
In zones with ZIKV circulation, pregnant women should be assessed for ZIKV exposure (with or without signs or symptoms). If there are fewer than 2 weeks of putative exposition, the recommended test is RT-PCR in both serum and urine samples. If these results are negative, an IgM ZIKV-ELISA should be performed 2–12 weeks later. If the pregnant woman visits the healthcare system 2–12 weeks after having symptoms or the putative exposure, the recommended test is ZIKV IgM ELISA with simultaneous testing of IgM to DENV. If both are positive, it means a recent flavivirus infection. In this case, it is necessary to evaluate antibody titres to each virus using a plaque reduction neutralization assay (PRNT). If the neutralization titres to ZIKV are >10, the diagnosis is a recent ZIKV infection [10].
In both confirmed and presumptive ZIKV infection during pregnancy, serial ultrasounds should be performed every 3–4 weeks to assess fetal anatomy and growth. Amniocentesis to evaluate fetal infection is not recommended. After birth, neonatal serum and urine should be tested by RT-PCR and IgM. If CSF is obtained for other reasons, it can also be tested. The placenta and umbilical cord, as well as tissues from fetal losses, can be processed for PCR and immunohistochemistry.
Conclusion
Emergent ZIKV is here to stay. Virus transmission can occur during the entire year because of the tropical weather and generalized A. aegypti infestation in developing countries. Because of the concurrent arbovirus epidemics and the overlapping endemic regions, the differential diagnosis must always include ZIKV, DENV and CHIKV. The development of new technical approaches to diagnose ZIKV infections and the clinical trials to evaluate them is an imperative need, mainly because of the deep impact on childbearing women in endemic zones.
References
1. Abushouka AI, Negidac A, Ahmed H. An updated review of Zika virus. J Clin Virol 2016; 84: 53–58.
2. Dos Santos T, Rodriguez A, Almiron M, Sanhueza A, Ramon P, et al. Zika virus and the Guillain–Barré syndrome – case series from seven countries. N Engl J Med 2016; 375(16): 1598–1601.
3. Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, et al. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis 2008; 14(8): 1232–1239.
4. Musso D, Roche C, Nhan TX, Robin E, Teissier A, Cao-Lormeau VM.Detection of Zika virus in saliva. J Clin Virol 2015; 68: 53–55.
5. Pardee K, Green AA, Takahashi MK, Braff D, Lamber G, et al. Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell 2016; 165: 1255–1266.
6. Faye O, Faye O, Dupressoir A, Weidmann M, Ndiaye M, Alpha Sall A. One-step RT-PCR for detection of Zika virus. J Clin Virol 2008; 43(1): 96–101.
7. Calvo EP, Sánchez-Quete F, Durán S, Sandoval I, Castellanos JE. Easy and inexpensive molecular detection of dengue, chikungunya and zika viruses in febrile patients. Acta Tropica 2016; 163: 32–37.
8. Musso D, Lanteri MC. Thoughts around the Zika virus crisis. Curr Infect Dis Rep 2016; 18(12): 46.
9. Steinhagen K, Probst C, Radzimski C, Schmidt-Chanasit J, Emmerich P, et al. Serodiagnosis of Zika virus (ZIKV) infections by a novel NS1-based ELISA devoid of cross-reactivity with dengue virus antibodies: a multicohort study of assay performance, 2015 to 2016. Euro Surveill 2016; 21(50): pii: 30426.
10. Centers for Disease Control and Prevention. Interim pregnancy guidance: testing and interpretation recommendations for a pregnant woman with possible exposure to Zika virus — United States (including U.S. territories). [https://www.cdc.gov/zika/pdfs/testing_algorithm.pdf]
The authors
Jaime E. Castellanos PhD, Shirly Parra-Álvarez and Eliana P. Calvo* PhD
Grupo de Virología, Universidad El Bosque, Bogotá, Colombia
*Corresponding author
E-mail: calvoeliana@unbosque.edu.co
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, /in Featured Articles /by 3wmediaClinical utility of PIVKA-II in the diagnosis of hepatocellular carcinoma
, /in Featured Articles /by 3wmediaPrimary liver cancer is the seventh most common cancer worldwide and the third most common cause of death from cancer [1]. Seventy-five to eighty-five percent of primary liver cancer cases are associated with HCC. The distinctive features of HCC include relatively large-sized tumours, vascular invasion, intra-hepatic metastasis, low differentiation, common recurrence and poor prognosis [2, 3]. In 70–90% of the cases, development of HCC requires a chronic liver disorder and cirrhosis as a background; these are caused mostly by chronic hepatitis C virus, hepatitis B virus, alcohol abuse, non-alcoholic steatohepatitis and less typically observed in inherited haemochromatosis, autoimmune hepatitis, antitrypsin deficiency, aflatoxin intoxication and also in some cases of oral contraception treatment [4, 5]. In addition, some chronic conditions, such as diabetes mellitus, cholelithiasis, obesity and hormone imbalance, are associated with HCC development [6]. The overall 5-year survival rate is believed to be less than 40%; however, a diagnosis at the early stages, followed by liver resection or transplantation, can improve this rate to 60–70% [7–9].
Taking into account the prevalence and mortality and also poor prognosis of HCC, it is apparent that highly sensitive techniques for diagnosis at the early stages are needed. The main diagnostic tool for HCC screening is radiologic imaging investigations such as ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI). With the development and introduction of contrast-enhanced ultrasound (CEUS) for analysis of intra-nodular vascularisation pattern, the sensitivity and specificity have been reported to be 90.9% and 100% for progressed HCC and 85.7% and 96.1% for early HCC, respectively [10]. The role of MRI and CT in producing reliable three-dimensional images is very important; however, the relationship between the radiographic and pathological tumour sizes is not yet well established. At this point application of tumour markers as supplementary analysis may provide useful information for making a diagnosis and monitoring of confirmed HCC [11, 12].
Protein induced by vitamin K absence/antagonist-II (PIVKA-II), also known as des-gamma carboxyprothrombin (DCP), is an abnormal form of prothrombin formed as a result of impaired or insufficient post-translational γ-carboxylation that occurs in the presence of vitamin K deficiency and leads to the loss of biological activity of the protein. Following synthesis in the liver, prothrombin, alongside the other hepatic vitamin K-dependent proteins undergoes transformation of specific glutamyl (GLU) residues into γ-carboxyl glutamyl (GLA) residues under the influence of vitamin K-dependent γ-glutamyl carboxylase in the presence of reduced vitamin K concentration (Fig. 1) [13]. Interestingly, carboxylation may not occur at all, which results in the formation of different variants of PIVKA-II with various degree of biological activity [14].
The role of PIVKA-II in HCC pathology is still not well established. It has been shown that PIVKA-II induces the malignant potential of HCC through stimulation of cell proliferation owing to a structural resemblance to hepatocyte growth factor [15–17]. Furthermore, PIVKA-II promotes angiogenesis in HCC resulting in local tissue invasion and metastases via stimulation of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) [13, 18].
Methods and patient samples
The automated chemiluminescent microparticle immunoassay (ARCHITECT PIVKA-II 2P4 CMIA, Abbott) was validated and used for quantitation of PIVKA-II using the Abbott™ Architect iSystem 2000 analyser in the Human Nutristasis Unit at St Thomas’ Hospital, London, UK. Imprecision and recovery evaluations were performed in line with the appropriate standard operating procedures as part of the validation process. The CMIA is based on a two-step sandwich reaction of binding of anti-PIVKA-II antibodies and specific PIVKA-II epitopes with subsequent addition of chemiluminescent labels and registration of the relative light units as a quantitative representation of PIVKA-II concentration in the tested sample [1].
In order to exclude possible interference with anticoagulant therapeutic agents, high PIVKA-II results were tested for warfarin, as it is the most commonly used anticoagulant that interferes with the vitamin K cycle. Samples found to be positive for warfarin were disqualified from further analysis.
Eighty-seven samples from the Gassiott Gastroenterology Clinic (GGC, St. Thomas’ Hospital, London) and the Hepatocellular Carcinoma Clinic in the Institute of Liver Studies (King’s College Hospital, London) were analysed in three groups: high-risk patients with non-HCC pathology of the liver, high-risk patients currently undergoing HCC surveillance, and patients with diagnosed HCC (group A, B and C respectively). Group A (n=29) consisted of randomly selected patients at GGC with viral and non-viral cirrhosis, steatosis, fibrosis, hepatitis and benign lesions. Group B (n=24) represented high-risk patients with changes to the liver suggestive of possible HCC discovered in the course of US/MRI/CT investigations. Finally, group C (n=34) comprised of patients diagnosed with HCC at different stages; the diagnosis was established in the course of histological examination of liver biopsy samples.
All results for PIVKA-II concentrations in patient samples were statistically processed in IBM SPSS Statistics, Version 23. Tests of normality, association between different variables and receiver operating characteristic (ROC) curve were applied for the analysis.
Results and discussion
Using a cut-off of 49.4 mAU/mL, an elevated PIVKA-II concentration was found in just one patient from the negative control group, which represents 3.4% (Table 1). This patient was diagnosed with multiple cysts on the background of hepatitis; therefore, the result may be interpreted as both false positive (elevation of PIVKA-II due to non-malignant pathology) and true positive (in this case the patient would need to undergo more comprehensive screening).
In the positive group, PIVKA-II was elevated in 79.4% of the patients and demonstrated a broad scatter of values (19.06 mAU/mL for the lowest detected concentration and 340 485.5 mAU/mL for the highest detected concentration) owing to various sizes of the tumour masses at different stages of HCC and possibly existence of different PIVKA-II variants depending on the number of GLU residues involved in γ-carboxylation [19]. Normal PIVKA-II results in this group can be explained by the normalisation of PIVKA-II concentration after curative treatment, if performed [16].
Statistical processing of data showed no evidence of dependence of the results on age or gender (P>0.05 for all three groups). Area under the curve (AUC) in ROC analysis for PIVKA-II in the present research was 0.917 (CI 95% 0.847–0.986), which is suggestive of excellent clinical usefulness of PIVKA-II in HCC diagnosis (Fig. 2). AUC for alpha-fetoprotein (AFP) had slightly lower value (0.833 with CI 95% 0.722–0.945), which can still be classified as a fairly useful test (Fig. 3).
In this study the optimal cut-off value for PIVKA-II was identified by means of ROC and is 49.4 mAU/mL with sensitivity of 79.4% and specificity of 96.6%. Analysis of true and false-negative and -positive results revealed, that more than 83% of PIVKA-II results were truly reliable, whereas only 74.6% of AFP results demonstrated true diagnostic value (Table 2).
Unfortunately, sensitivity and specificity of AFP cannot accurately reflect its performance in the present study, as AFP results were available for only 17 patients from group A, which means that the study was possibly deprived of some potentially truly negative results. However, taking into account considerable difference between sensitivity and specificity rates for PIVKA-II and AFP (79.4 vs 96.6% and 70.6 vs 82.4% respectively), allows the conclusion that PIVKA-II displays slightly better clinical utility in HCC diagnosis. Similar results were reported in the previous studies [7, 20–24].
Limitations to the study
The major limitation to this research was the requirement to use anonymised samples, which prevented access to the full clinical history of the patients and impossibility to interpret the results in detail. Another limitation was the number of samples which could be considered to be insufficient to achieve aims of the project with adequate statistical power. A larger number of samples would have given the study more power and allowed a more precise ROC to be constructed and subsequently a more precise cut-off value to be identified.
Conclusion
In the present research PIVKA-II demonstrated high accuracy, sensitivity and specificity in HCC diagnosis. PIVKA-II has several advantages over AFP in terms of clinical utility for HCC diagnosis and prognosis: PIVKA-II is comparatively less frequently elevated in liver pathology [22], is more sensitive to small HCC tumours, correlates with HCC progression significantly better and has shorter half-life than AFP (40–72 hours against 5–7 days), which makes it more suitable for monitoring purposes [14]. Implementation of PIVKA-II as diagnostic test gathers pace in transplantation medicine, as this tumour marker, alongside Milan criteria has been used for recipient selection for living donor liver transplantation [16]. In addition, PIVKA-II concentrations can reflect the responsiveness of the liver to medical treatment (i.e. sorafenib), which cannot be achieved with AFP test. On the other hand, AFP is sensitive to radiological response following transarterial chemoembolisation, whereas PIVKA-II is not [12]. Also, PIVKA-II is affected by potentially interfering pharmacological agents (e.g. warfarin and certain antibiotics), it is dependent on vitamin K metabolism and can give false-positive results in non-HCC conditions which all has to be taken into account while interpreting the results.
Controversy over the best performance of tumour markers traces back to different assays used and various patient groups involved. Fortunately, AFP and PIVKA-II are independent of each other [16, 25]. Therefore, combination of PIVKA-II and AFP alongside AFP-L3, the fucosylated fraction of AFP, is suggested to be the best option for highly accurate laboratory diagnostic of HCC supplementary to imaging techniques. This multi-marker approach has been stated in the guidelines of The Japan Society of Hepatology and successfully used for diagnosis and management of HCC in Japan [26, 27].
Acknowledgement
ARCHITECT PIVKA-II 2P4 CMIA reagents and the graphics used in this article are courtesy of © Abbott Laboratories.
References
1. Kinukawa H, et al. characterization of an anti-PIVKA-II antibody and evaluation of a fully automated chemiluminescent immunoassay for PIVKA-II. Clin Biochem 2015; 48: 1120–1125.
2. Ha TY, et al. Expression pattern analysis of hepatocellular carcinoma tumour markers in viral hepatitis B and C patients undergoing liver transplantation and resection. Transplant Proc 2014; 46: 888–893.
3. Yano Y, et al. Clinical features of hepatitis C virus-related hepatocellular carcinoma and their association with α-fetoprotein and protein induced by vitamin K absence or antagonist-II. Liver Int 2006; 26: 789–795.
4. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007; 132: 2557–2576.
5. Aghemo A, Colombo M. Hepatocellular carcinoma in chronic hepatitis C: from bench to bedside. Semin Immunopathol 2012; 35: 111–120.
6. McMasters K, Vauthey J. Hepatocellular carcinoma: targeted therapy and multidisciplinary care. Springer 2011; Chapters 1–5, 8.
7. Ji J, et al. Diagnostic evaluation of des-gamma-carboxy prothrombin versus α-fetoprotein for hepatitis B virus-related hepatocellular carcinoma in China: a large-scale, multicentre study. PLoS One 2016; 11: e0153227.
8. Huang TS, et al. Diagnostic performance of alpha-fetoprotein, lens culinaris agglutinin-reactive alpha-fetoprotein, des-gamma carboxyprothrombin, and glypican-3 for the detection of hepatocellular carcinoma: a systematic review and meta-analysis protocol. Syst Rev 2013; 2: 37.
9. Song PP, et al. Controversies regarding and perspectives on clinical utility of biomarkers in hepatocellular carcinoma. World J Gastroenterol 2016; 22: 262–274.
10. Giorgio A, et al. Characterization of dysplastic nodules, early hepatocellular carcinoma and progressed hepatocellular carcinoma in cirrhosis with contrast-enhanced ultrasound. Anticancer Res 2011; 31: 3977–3982.
11. Chen H, et al. CT and MRI in target delineation in primary hepatocellular carcinoma. Cancer Radiother 2013; 17: 750–754.
12. Park H, Park JY. Clinical significance of AFP and PIVKA-II responses for monitoring treatment outcomes and predicting prognosis in patients with hepatocellular carcinoma. BioMed Research International 2013; 2013: 310427.
13. Yue P, et al. Des-γ-carboxyl prothrombin induces matrix metalloproteinase activity in hepatocellular carcinoma cells by involving the ERK1/2 MAPK signalling pathway. Eur J Cancer 2011; 47: 1115–1124.
14. Zhang YS, et al. Des-γ-carboxy prothrombin (DCP) as a potential autologous growth factor for the development of hepatocellular carcinoma. Cell Physiol Biochem 2014; 34: 903–915.
15. Suzuki K, et al. Positioning of novel tumor marker NX-PVKA-R in the diagnosis of hepatocellular carcinoma in comparison with PIVKA-II. Dokkyo Journal of Medical Sciences 2013; 40: 163–168
16. Inagaki Y, et al. Clinical and molecular insights into the hepatocellular carcinoma tumour marker des-γ-carboxyprothrombin. Liver Int 2010; 31: 22–35.
17. Jinghe X, et al. Vitamin K and hepatocellular carcinoma: the basic and clinic. World J Clin Cases 2015; 3: 757–764.
18. Fujikawa T, et al. Significance of des-gamma-carboxyprothrombin production in hepatocellular carcinom. Acta Med Okayama 2009; 63: 299–304.
19. Zakhary NI, et al. Impact of PIVKA-II in diagnosis of hepatocellular carcinoma. J Adv Res 2013; 4: 539–546.
20. Mathew S, et al. Biomarkers for virus-induced hepatocellular carcinoma (HCC). Infect Genet Evol 2014; 26: 327–339.
21. Lim TS, et al. Combined use of AFP, PIVKA-II, and AFP-L3 as tumor markers enhances diagnostic accuracy for hepatocellular carcinoma in cirrhotic patients. Scand J Gastroenterol 2015; 51: 344–353.
22. Seo SI, et al. Diagnostic value of PIVKA-II and alpha-fetoprotein in hepatitis B virus-associated hepatocellular carcinoma. World J Gastroenterol 2015; 21: 3928–3935.
23. De J, et al. A systematic review of des-γ-carboxy prothrombin for the diagnosis of primary hepatocellular carcinoma. Medicine 2016; 95: e3448.
24. Ette AI, et al. Utility of serum des-gamma-carboxyprothrombin in the diagnosis of hepatocellular carcinoma among Nigerians, a case–control study. BMC Gastroenterol 2015; 15: 113.
25. Choi JY, et al. Diagnostic value of AFP-L3 and PIVKA-II in hepatocellular carcinoma according to total-AFP. World J Gastroenterol 2013; 19: 339–346.
26. Kudo M. Clinical practice guidelines for hepatocellular carcinoma differ between Japan, United States, and Europe. Liver Cancer 2015; 4: 85–95.
27. Kokudo M, et al. Evidence-based clinical practice guidelines for hepatocellular carcinoma: The Japan Society of Hepatology 2013 update (3rd JSH-HCC Guidelines). Hepatol Res 2015; 45: 123–127.
The authors
Volha Klimovich*1 MSc; Kieran Voong2 MSc; Roy Sherwood3 MSc, DPhil; Dominic J Harrington2 MSc, PhD
1Clinical Biochemistry, Viapath, St Thomas’ Hospital, London, UK
2Human Nutristasis Unit, Viapath, St Thomas’ Hospital, London, UK
3Viapath, King’s College Hospital, London, UK
*Corresponding author
E-mail: klimovichvolha@gmail.com
Improving diagnosis of Zika virus infection: an urgent task for pregnant women
, /in Featured Articles /by 3wmediaZika virus (ZIKV) belongs to the Flavivirus genus and is related to other viruses that are also transmitted by the bite of mosquitoes, such as dengue virus (DENV), yellow fever virus (YFV) and West Nile virus (WNV). The Flaviviridae family comprises single-strand RNA, membrane-enveloped viruses that frequently use Aedes aegypti as a vector. Despite ZIKV being discovered over 60 years ago, only since 2014 (in the French Polynesia Islands) and 2015 (Brazil and America) has it been evident that the virus can cause large outbreaks and epidemics that lead to a global public health emergency [1].
ZIKV infection causes a mild severity, undifferentiated febrile syndrome, characterized by rash, arthralgia, myalgia and conjunctivitis, symptoms that are similar to those that appear in DENV fever or chikungunya virus (CHIKV) fever (CHIKV being an unrelated alphavirus transmitted by the same mosquito). The similarities of the symptoms causes confusion between the diseases during clinical evaluation. Also, these three viral illnesses may co-circulate in the same areas, hampering the final diagnosis of patients.
Although the ZIKV morbidity and mortality are considered low, it was demonstrated during the recent outbreaks that infection in pregnant women may be associated with severe birth defects (mostly microcephaly), and with the appearance in infected adults of a severe neurologic disease called Guillain–Barré syndrome (GBS). This neurologic entity increased 2–10-fold the historic cases in Latin America during the 2016 ZIKV epidemic [2]. Epidemiological estimates consider that approximately 75% of ZIKV-infected people do not present signs or symptoms during an outbreak, but they become an efficient transmission focus to mosquitoes and other individuals.
It is well known that mosquito bites are the main transmission route in areas where the insect infestation rates are high; however, it recently has been confirmed that ZIKV is capable of crossing the placental barrier and infecting the fetus. In adult patients, the virus persists in semen and vaginal fluids for two months, producing a viral load sufficient for transmission during sexual intercourse. This finding changes the epidemiological trends, as it is now also possible to detect infected patients in non-tropical countries, challenging the clinical and laboratory diagnosis. However, it is clear that tropical underdeveloped countries will still be the major source of febrile cases and, of course, the congenital malformations and GBS appearance in adults.
ZIKV infection diagnostics
The incubation period of ZIKV disease is not clear but is likely to be a few days, similar to other arboviruses. Symptoms can begin 2 to 7 days after a mosquito bite and last for 3 to 7 additional days. In both early symptomatic or asymptomatic cases, the virus can be detected by reverse transcription (RT)-PCR after purification of plasma or serum RNA. The acute sera can be inoculated in Vero cells or C6/36 mosquito cells to attempt virus isolation, but although this technique is powerful, it is expensive and lacks clinical value. We successfully isolated ZIKV and produced enough inoculum for cell biology and immunologic studies (Fig. 1). As a result of their sensitivity and specificity, ZIKV RNA detection by different nucleic acid tests is used on a routine basis to confirm acute ZIKV cases.
RT-PCR
The real-time RT-PCR protocol designed by the Centers for Disease Control (CDC, USA) during the 2007 Yap Island outbreak is the most used and evaluated, even after the confirmation that a very low viral load occurs during the acute phase and that viremia lasts only a few days in both symptomatic and asymptomatic individuals. This CDC protocol does not amplify RNA from other flaviviruses and alphaviruses [3].
The test was designed as a one-step RT-PCR with fluorogenic probes using serum as the sample and is also used on urine samples, where the virus can be detected until 15 days after symptoms start and when the serum sample has become negative. A comparison between different sample types demonstrated that saliva may be better than serum for confirming ZIKV infection [4]. A very sensitive and specific synthetic biology tool based on isothermal amplification and toehold switch RNA sensors has been reported and is currently under evaluation in field conditions in Colombia, Brazil and Equator [5].
Many other real-time PCR tests have recently been developed, but there are no reports regarding their clinical evaluation. One test with excellent analytical performance is becoming available (Altona Diagnostics), but it has not yet reported clinical assays in ZIKV circulating zones.
Frequently, conventional PCR has been used to follow epidemics and ZIKV circulation in mosquitoes [6], and this reported test was used to confirm the first cases in Brazil. Recently, we used modified primers to perform a double-round one-step RT-PCR to detect DENV, ZIKV and CHIKV in the serum of febrile patients, obtaining samples simultaneously positive for two or even three viruses [7]. This test also detects ZIKV RNA in paired samples of serum, breast milk and urine (Fig. 2).
Serology
The main challenge to serological ZIKV diagnosis is related to its structural proximity to other flaviviruses (DENV, YFV, and WNV) because antibodies against one of them can recognize the other viruses on ELISA platforms, frequently resulting in a false positive diagnostic. For this reason, RNA detection is preferred to confirm the infection during the first week after symptoms appear. However, serological tests are recommended to facilitate the diagnosis of pregnant women living in endemic zones or women or couples wanting pre-conception counselling because ZIKV IgM positivity confirms previous exposure to the virus; in those who are negative, it is recommended to perform periodic tests to prove the absence of virus contact.
Since the first serological diagnostics were performed in Africa, there have been difficulties confirming the infection by antibodies due to cross-reactivity in neutralization tests, hemagglutination inhibition and mouse neutralization used in the 1950s [8]. During the Yap Islands’ 2007 ZIKV outbreak, in addition to molecular confirmation, 14 ZIKV cases were investigated by serology, and it was confirmed that 8/14 individuals who had a previous flavivirus infection (secondary flavivirosis) were positive by DENV IgM ELISA. In addition, ZIKV-confirmed sera had high titres in the plaque reduction neutralization test (PRNT), mainly to DENV (12/14), YFV (3/14) and WNV (6/14) [3].
Currently, the CDC uses IgM antibody capture (MAC)-ELISA in its diagnostic algorithm, in which the ZIKV antigen is obtained from infected mice brains or recombinant proteins (Fig. 3a). This test is being used to confirm recent infections and to counsel women in endemic zones. This ELISA has not yet been tested in endemic zones where other flaviviruses circulate. Recently, an assay based on non-structural protein NS1 from ZIKV adsorbed to ELISA plates has been reported (Euroimmun AG), showing excellent performance to detect both IgM and IgG using samples from endemic zones and samples with confirmed contact with other flaviviruses (Fig. 3b) [9].
Pregnant women: the priority
Considering that ZIKV infection during the first two trimesters of pregnancy can be associated with neurological defects in the fetus, it is important to evaluate the infection risk in three different groups of women: (i) women of childbearing age living in areas with virus circulation; (ii) women travelling frequently to endemic zones; and (iii) women having sexual intercourse with individuals travelling frequently to ZIKV endemic zones (Table 1). Notably, only 25% of infected individuals present signs or symptoms of the disease, but those who are asymptomatic can transmit the virus to mosquitoes and through sexual contact, can develop GBS or can transmit the virus to the fetus during pregnancy. Normally, health authorities do not recommend confirming all cases by PCR or serology, but only those needed to facilitate the surveillance of ZIKV infections or sequelae (Table 1).
CDC testing algorithm
In zones with ZIKV circulation, pregnant women should be assessed for ZIKV exposure (with or without signs or symptoms). If there are fewer than 2 weeks of putative exposition, the recommended test is RT-PCR in both serum and urine samples. If these results are negative, an IgM ZIKV-ELISA should be performed 2–12 weeks later. If the pregnant woman visits the healthcare system 2–12 weeks after having symptoms or the putative exposure, the recommended test is ZIKV IgM ELISA with simultaneous testing of IgM to DENV. If both are positive, it means a recent flavivirus infection. In this case, it is necessary to evaluate antibody titres to each virus using a plaque reduction neutralization assay (PRNT). If the neutralization titres to ZIKV are >10, the diagnosis is a recent ZIKV infection [10].
In both confirmed and presumptive ZIKV infection during pregnancy, serial ultrasounds should be performed every 3–4 weeks to assess fetal anatomy and growth. Amniocentesis to evaluate fetal infection is not recommended. After birth, neonatal serum and urine should be tested by RT-PCR and IgM. If CSF is obtained for other reasons, it can also be tested. The placenta and umbilical cord, as well as tissues from fetal losses, can be processed for PCR and immunohistochemistry.
Conclusion
Emergent ZIKV is here to stay. Virus transmission can occur during the entire year because of the tropical weather and generalized A. aegypti infestation in developing countries. Because of the concurrent arbovirus epidemics and the overlapping endemic regions, the differential diagnosis must always include ZIKV, DENV and CHIKV. The development of new technical approaches to diagnose ZIKV infections and the clinical trials to evaluate them is an imperative need, mainly because of the deep impact on childbearing women in endemic zones.
References
1. Abushouka AI, Negidac A, Ahmed H. An updated review of Zika virus. J Clin Virol 2016; 84: 53–58.
2. Dos Santos T, Rodriguez A, Almiron M, Sanhueza A, Ramon P, et al. Zika virus and the Guillain–Barré syndrome – case series from seven countries. N Engl J Med 2016; 375(16): 1598–1601.
3. Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, et al. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis 2008; 14(8): 1232–1239.
4. Musso D, Roche C, Nhan TX, Robin E, Teissier A, Cao-Lormeau VM.Detection of Zika virus in saliva. J Clin Virol 2015; 68: 53–55.
5. Pardee K, Green AA, Takahashi MK, Braff D, Lamber G, et al. Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell 2016; 165: 1255–1266.
6. Faye O, Faye O, Dupressoir A, Weidmann M, Ndiaye M, Alpha Sall A. One-step RT-PCR for detection of Zika virus. J Clin Virol 2008; 43(1): 96–101.
7. Calvo EP, Sánchez-Quete F, Durán S, Sandoval I, Castellanos JE. Easy and inexpensive molecular detection of dengue, chikungunya and zika viruses in febrile patients. Acta Tropica 2016; 163: 32–37.
8. Musso D, Lanteri MC. Thoughts around the Zika virus crisis. Curr Infect Dis Rep 2016; 18(12): 46.
9. Steinhagen K, Probst C, Radzimski C, Schmidt-Chanasit J, Emmerich P, et al. Serodiagnosis of Zika virus (ZIKV) infections by a novel NS1-based ELISA devoid of cross-reactivity with dengue virus antibodies: a multicohort study of assay performance, 2015 to 2016. Euro Surveill 2016; 21(50): pii: 30426.
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The authors
Jaime E. Castellanos PhD, Shirly Parra-Álvarez and Eliana P. Calvo* PhD
Grupo de Virología, Universidad El Bosque, Bogotá, Colombia
*Corresponding author
E-mail: calvoeliana@unbosque.edu.co
Simple, fast and highly sensitive colorimetric detection of Zika virus
, /in Featured Articles /by 3wmediaBackground
An outbreak of Zika virus (ZIKV) in Brazil terrorized the whole world and its explosive spread in the Americas caused the World Health Organization (WHO) to declare it a public health emergency of international concern in February 2016 [1]. This is because ZIKV is a suspected major cause of congenital microcephaly, Guillain-Barré syndrome and other neurologic syndromes [2–4]. ZIKV has a genome consisting of a single-stranded, positive-polarity RNA and belongs to the family Flaviviridae and the genus Flavivirus. Aedes mosquitoes, known as a major ZIKV vector, also transmit dengue and chikungunya viruses across tropical and subtropical regions around the world [5]. Moreover, antigenic similarity between ZIKV and dengue virus gives rise to serological cross-reactivity, precluding antibody-based assays from reliably distinguishing between ZIKV and dengue virus infections [6]. Thus, reliable methods for distinguishing ZIKV from dengue and chikungunya viruses are necessary in practical applications.
WHO target product profiles
In April 2016, the WHO announced Target Product Profiles (TPPs) for a better diagnostic test for ZIKV infection. The TPPs define the desired characteristics of a ZIKV diagnostic test. The proposed TPPs consist of ‘Detection of active infection with ZIKV’ (Table 1a) and ‘Detection of evidence of prior infection’ (Table 1b). Each characteristic in the tables represents essential properties that the newly developed ZIKV diagnostic test should have at least at an acceptable level. To state the obvious, the criteria of specificity for active infection are more stringent [7].
Previous research on ZIKV diagnostics
Due to serological cross-reactivity between ZIKV and other flaviviruses, most of previous studies on ZIKV diagnosis have dealt with molecular diagnostics instead of immunological assays. Faye and colleagues developed and evaluated a one-step reverse transcription (RT)-PCR assay for ZIKV detection. The limit of detection of the assay was found to be 7.7 plaque-forming units (p.f.u.) per reaction in human serum and in the L-15 medium [8]. A quantitative real-time RT-PCR assay for ZIKV was also developed by the same research group. Analytical sensitivity of the assay was estimated at 3.2×102 RNA copies/μL [9]. However, a conventional PCR assay requires a bulky and expensive thermal cycler, prolonged reaction time, and trained technicians; these resources are not available in many low- and middle-income countries. Moreover, the RT-PCR reaction is vulnerable to inhibitors (blood, plasma and urine), thus requiring painstaking and cumbersome RNA extraction steps.
Recent research on ZIKV diagnostics
To overcome such limitations of RT-PCR, a variety of isothermal nucleic acid amplification techniques have recently been developed. Among them, reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid, robust, and highly sensitive isothermal RNA amplification method that uses four to six primers to amplify specific RNA sequences at 60–65°C even in the presence of inhibitors such as blood, plasma, or urine. RT-LAMP is much faster than conventional PCR, and the reaction can even proceed in an oven, water bath or with heating packs [10, 11]. Despite these advantages, the RT-LAMP assays still rely on a conventional bulky amplicon analyser such as a gel electrophoresis apparatus or a fluorescence laser-induced detector for monitoring the LAMP amplicons; this situation precludes the use of RT-LAMP in point-of-care diagnosis.
Our approaches to simple and highly sensitive diagnosis of ZIKV
To eliminate the dependence on a conventional amplicon analyser while retaining the aforementioned advantages of RT-LAMP, we selected the lateral flow assay (LFA) format for RT-LAMP amplicon analysis. The LFA, a driving principle behind pregnancy test strips, is also widely known as a superior diagnostic tool for nucleic acids owing to its high sensitivity, simplicity, selectivity and easy interpretation of results. Moreover, the Bst 3.0 polymerase used in this study for RT-LAMP retains both improved isothermal amplification performance and strong reverse transcription activity, allowing us to avoid addition of exogenous reverse transcriptase and the inhibition of reverse transcription by biological substances. By utilizing the advantages of Bst 3.0 polymerase and combining the RT-LAMP assay with the LFA, we demonstrated simple and highly sensitive detection of ZIKV RNA in human whole blood by merely observing a colorimetric signal within 35 min.
The RT-LAMP reaction and modification of amplicons in our study
As mentioned above, RT-LAMP has excellent tolerance to many inhibitors so that isothermal amplification of ZIKV RNA is possible even when human whole blood is directly used as a sample. We extracted ZIKV RNA and added it into human whole blood to mimic ZIKV-containing blood samples. Then, the spiked human whole blood was serially diluted with blood to set up a concentration range from 106 copies of RNA to a single copy per 2 μL and directly used these dilutions as samples without additional RNA purification steps. To colorimetrically detect the result of the LFA, a special modification is needed: labelling of the amplicon with digoxigenin and biotin. Among our own designed ZIKV-specific primers, two loop primers were tagged with digoxigenin at the 5´end; this approach will allow digoxigenin to label the amplicon when loop primers amplify the ZIKV RNA by the LAMP method. Labelling of the amplicon with biotin is made possible by adding biotin-labelled dUTP (Biotin-dUTP) to the mix of deoxynucleotides (dNTPs) at a certain ratio. When ZIKV RNA is amplified and this reaction consumes dNTPs, Biotin-dUTP will substitute thymine at the adenine sites of the complementary strand, resulting in labelling of the amplicon with biotin.
RT-LAMP was carried out in a 25 μL reaction mixture containing 1× Isothermal Amplification Buffer II [20 mM Tris-HCl, 10 mM (NH4)2SO2, 150 mM KCl, 2 mM MgSO4, and 0.1% Tween 20], additional 2 mM MgSO4, a dNTP mix supplemented with biotin-dUTP (2.2 mM dGTP, dATP, dCTP, 1.375 mM dTTP, and 0.0825 mM biotin-dUTP), a target-specific primer mixture (0.8 μM forward and reverse inner primers, 0.4 μM digoxigenin-labelled loop primers, and 0.2 μM forward and reverse outer primers), 8 U of Bst 3.0 DNA polymerase, and 2 μL of human whole blood spiked with ZIKV RNA ranging from 106 copies to a single copy per 2 μL. The RT-LAMP reaction mixture was incubated for 30 min.
Design and operation of the LFA
Figure 1(a) and 1(b) shows the detailed set-up and operating procedures of the LFA in our study. First, 1 μL of digoxigenin- and biotin-labelled RT-LAMP products was loaded onto the conjugate pad, so that the biotin-labelled RT-LAMP products formed a complex with gold nanoparticles (AuNPs) via streptavidin-biotin interactions. Next, 45 μL of diluent buffer was placed on the buffer loading pad, and then capillary flow transferred AuNPs from the conjugate pad to the test and control line. The AuNP–RT-LAMP complexes were immobilized at the test line by the interaction between digoxigenin and anti-digoxigenin whereas the AuNPs that did not form complexes were captured by biotin. Complexed and uncomplexed AuNPs are indicated by violet bands at the test line and control line, respectively. The colorimetric signal was easily visible with the naked eye within 5 min.
Discussion
Analysis of the limit of detection in human whole blood samples
We evaluated the limit of detection of the LFA to determine whether our method is indeed highly sensitive. Two microliters of human whole blood was directly used as a sample without any purification steps. Figure 1(c) shows the ZIKV RNA detection results for the LFA. The signal intensities on the test line gradually declined as the concentration of ZIKV RNA decreased. Notably, the presence of even a single copy of ZIKV RNA could be detected within 35 min by the LFA. These results imply that our method has a great potential for diagnosis of ZIKV infections.
References
1. Lessler J, Chaisson LH, Kucirka LM, Bi Q, Grantz K, Salje H, et al. Assessing the global threat from Zika virus. Science 2016; 353: aaf8160.
2. Schuler-Faccini L, Ribeiro E, Feitosa I, Horovitz D, Cavalcanti D, et al. Possible Association Between Zika Virus Infection and Microcephaly Brazil, 2015. MMWR Morb Mortal Wkly Rep 2016; 65: 59–62.
3. Cao-Lormeau V-M, Blake A, Mons S, Lastère S, Roche C, et al. Guillain-Barré Syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. Lancet 2016; 387: 1531–1539.
4. WHO statement on the first meeting of the International Health Regulations (2005) (IHR 2005) Emergency Committee on Zika virus and observed increase in neurological disorders and neonatal malformations. http://www.who.int/mediacentre/news/ statements/2016/1st-emergency-committee-zika/en/ (accessed May 1).
5. Surveillance and Control of Aedes aegypti and Aedes albopictus in the United States. http://www.cdc.gov/chikungunya/resources/ vector-control.html (accessed May 1).
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7. Target product profiles for better diagnostic tests for Zika virus infection. http://www.who.int/csr/research-and-development/zika-tpp.pdf.
8. Faye O, Faye O, Dupressoir A, Weidmann M, Ndiaye M, Alpha Sall A. One-step RT-PCR for detection of Zika virus. J Clin Virol 2008; 43: 96–101.
9. Faye O, Faye O, Diallo D, Diallo M, Weidmann M, Sall AA. Quantitative real-time PCR detection of Zika virus and evaluation with field-caught mosquitoes. Virol J 2013; 10: 311.
10. Safavieh M, Kanakasabapathy MK, Tarlan F, Ahmed MU, Zourob M, et al. Emerging loop-mediated isothermal amplification-based microchip and microdevice technologies for nucleic acid detection. ACS Biomater Sci Eng 2016; 2: 278–294.
11. Nyan D-C, Ulitzky LE, Cehan N, Williamson P, Winkelman V, et al. Rapid detection of hepatitis B virus in blood plasma by a specific and sensitive loop-mediated isothermal amplification assay. Clin Infect Dis 2014; 59: 16–23.
The authors
Dohwan Lee MS, Yong Kyoung Yoo PhD, and Jeong Hoon Lee* PhD
Department of Electrical Engineering, Kwangwoon University, Nowon, Seoul 01897, Republic of Korea.
*Corresponding author
E-mail: jhlee@kw.ac.kr
Point-of-care molecular test for Zika infection
, /in Featured Articles /by 3wmediaThe recent emergence of the Zika virus (ZIKV) pandemic has underscored the need for a point-of-care (POC) test that can detect viral infections, fostering widespread, accurate and timely diagnostics [1, 2]. Zika infection is often asymptomatic or has comparatively mild symptoms, such as fever and chills that are common to many other infections. However, as is widely known, Zika infection during pregnancy entails substantial risks for the newborn, including severe birth defects [3]. Hence, it is highly desirable to screen women of child-bearing age and their partners for exposure to the ZIKV. Zika episodes have created bottlenecks in conventional laboratory-based diagnostic.
Zika is spread primarily by mosquitos, but sexual and perinatal transmission, as well as transmission via blood transfusions have also been reported. Specific applications for Zika diagnostics thus include determining whether infection has occurred over the course of a pregnancy, whether sexual partners harbour infection, safeguarding the blood supply and tracking the geographic range of Zika infections [4]. In order to inform investment decisions on prevention, control and response, Lee et al. [5] modelled the economic burden for various scenarios of Zika emergence across six US states, estimating total costs (direct medical, Medicaid, productivity losses) ranging from 0.5 to 2 billion US dollars.
Ideally, a POC technology will enable rapid diagnostics tests that can be performed outside of centralized laboratories such as, for example, in doctors or dentists offices, pharmacies, rural clinics, school infirmaries, border crossings or, ultimately, as an over-the-counter test for home use. Thus, the availability of a mass-produced, inexpensive POC diagnostics device with easy-to-interpret test results, and that could be used in any almost any locale by non-specialists with minimal training, would greatly expand capabilities and options for screening, surveillance, diagnostics, monitoring and therapy.
Current diagnostics technology
According to the World Health Organization (WHO), the pipeline diagnostic ZIKV kits can be categorized into: (i) antibody/antigen-based immunoassay and (ii) nucleic acid-based molecular diagnostics [6]. Immunoassay (antibody detection) for ZIKV infection utilizes envelope proteins and NS1 as targets. The major challenge is that these antibodies cross-react with other highly homologous flaviviruses such as dengue, resulting in non-specific test results [7]. IgM and IgG antibodies, typically emerge, respectively, ~4 and ~10 days after infection, but are usually undetectable until >7–14 days post-infection. Moreover, antibody responses during pregnancy may differ from those in non-pregnant individuals [8], which may adversely impact the effectiveness of immunoassay tests. Moreover, antibody tests may not discriminate between recent and historic exposure. The Food and Drug Administration (FDA) recently authorized for emergency use of the IgM Antibody Capture Enzyme-Linked Immunosorbent Assay (Zika MAC-ELISA) to detect ZIKV [8]. However, this assay requires a lab-format with delays in generating results given current demand. More importantly, these assays are a readout for exposure to ZIKV, whereas active virus infection is not determined. Currently, several companies are developing lateral flow-based rapid diagnostic test for ZIKV antibody detection [9, 10].
Molecular diagnostics-based on reverse-transcription (RT)-PCR is highly specific and sensitive, and considered the gold standard for ZIKV detection. RT-PCR is effective in serum, semen, and saliva within 14 days post-infection, and possibly much longer in urine and semen [11, 12]. Importantly, a recent study has demonstrated that ZIKV is detectable in pregnant women throughout their pregnancy [3]. Indeed, the FDA has authorized the use of the Trioplex rRT-PCR laboratory test to detect ZIKV, dengue virus, and chikungunya virus RNA, under an Emergency Use Authorization (EUA) [13]. Several research groups and companies are developing multiplexed molecular assays to concurrently detect various members of the genus Flavivirus. Most of these RT-PCR kits require, however, instrumentation and are for central laboratory use only.
Instrument-free point-of-care molecular detection of ZIKV
To develop inexpensive molecular detection of ZIKV without complex instrumentation, we utilized reverse-transcription loop-mediated amplification (RT-LAMP) technology [14]. We identified highly conserved regions of the ZIKV genome and designed RT-LAMP primers for the Zika lineage that is prevalent in the Americas. To enable POC molecular diagnostics, we developed a disposable microfluidic cassette (Fig. 1a) that combines viral nucleic acid capture, concentration, isothermal amplification; and detection. Our disposable, microfluidic cassette contains multiple independent amplification reactors, each equipped with a silica-based nucleic acid isolation membrane at its inlet. The advantage of such a design is to decouple the sample volume from the reaction volume, allowing one to use relatively high sample volumes to achieve high sensitivity. Nucleic acids captured by the isolation membrane directly serve as templates in an RT-LAMP amplification process without a need for an elution step, significantly simplifying flow control.
Our microfluidic cassette can be incubated with battery power or operate electricity-free. To eliminate the need for electricity, we used a simple, thermally insulated portable cup (Fig. 1b) heated by an exothermic reaction for chip-based isothermal amplification [14]. One Mg−Fe alloy pouch, which is usually used as a heater of MRE (meal, ready-to-eat), served as the heat source. Tap water was introduced into the drawer, housing the Mg-Fe pouch, through a port in the cup lid to interact with the Mg−Fe alloy to produce heat. To isolate the amplification reactor’s temperature from variable ambient conditions, we used a phase change material (PCM) to regulate the temperature, removing the need for a thermal control circuit. An aluminium heat sink was used to enhance heat transfer from the PCM to the cassette.
We tested the utility of our POC diagnostic system with raw saliva samples spiked with various concentrations of the ZIKV. Our experiments showed that our electricity-free POC diagnostic system could detect ZIKV in saliva with the sensitivity of 5 plaque forming units (p.f.u.) of ZIKV per sample within 40 min (Fig. 1c). Our POC diagnostic system is comparable to that of the benchtop assay without a need for laboratory facilities, expensive equipment and well-trained personnel [14].
Conclusion
Zika molecular diagnostics can be performed at the point of care with a low-cost, portable system based on a microfluidic cassette that integrates nucleic acid isolation and concentration, isothermal amplification, and detection. To achieve electricity-free isothermal amplification, the cassette is combined with a chemically heated cup that generates heat with an exothermic reaction. The platform can be adapted to various sample types and sizes, and multiplex detection. This flexibility is useful in view of the evolving understanding of Zika pathology and Zika biomarker levels and their persistence in different body fluids and tissues. In the future, we plan to expand system capabilities to enable concurrent detection of multiple vector-borne diseases [15]. Our system is very suitable for resource-poor settings, where funds, centralized laboratory facilities and trained personnel are in short supply, as well as for use in remote clinics and at home.
Acknowledgment
The research reported here was supported, in part by the NIH NIDCR R21DE026700, K25AI099160, R01 CA214072, to the University of Pennsylvania.
References
1. Cao-Lormeau VM, Blake A, Mons S, Lastère S, Roche C, et al. Guillain-Barré Syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. Lancet 2016; 387(10027): 1531–1539.
2. Tang H, Hammack C, Ogden SC, Wen Z, Qian X, et al. Zika virus infects human cortical neural progenitors and attenuates their growth. Cell Stem Cell 2016; 18(5): 587–590.
3. Driggers RW, Ho CY, Korhonen EM, Kuivanen S, Jääskeläinen AJ, et al. Zika virus infection with prolonged maternal viremia and fetal brain abnormalities. N Eng J Med 2016; 374: 2142–2151.
4. Kindhauser MK, Allen T, Frank V, Santhana RS, Dye C. Zika: the origin and spread of a mosquito-borne virus. Bull World Health Organ 2016; 94: 675–686C.
5. Lee BY, Alfaro-Murillo JA, Parpia AS, Asti L, Wedlock PT, et al. The potential economic burden of Zika in the continental United States. PLOS Neg Trop Dis 2017; 11(4): e0005531.
6. Current Zika product pipeline. World Health Organization (WHO) 2016. http://www.who.int/csr/research-and-development/zika-rd-pipeline.pdf.
7. Charrel, RN, Leparc-Goffart I, Pas S, de Lamballerie X, Koopmans M, Reusken C. State of knowledge on Zika virus for an adequate laboratory response. Bull World Health 2016; 94: 574–584D.
8. New CDC laboratory test for Zika virus authorized for emergency use by FDA. Centers for Disease Control and Prevention (CDC) 2016. https://www.cdc.gov/media/releases/2016/s0226-laboratory-test-for-zika-virus.html.
9. Zika rapid test. Biocan Diagnostics Inc. http://www.zikatest.com/?page_id=6.
10. Artron Zika test. Artron Laboratories Inc. http://www.artronlab.com/home.html.
11. Gourinat AC, O’Connor O, Calvez E, Goarant C, Dupont-Rouzeyrol M. Detection of Zika virus in urine. Emerg Infect Dis 2015; 21(1): 84.
12. Mansuy JM, Dutertre M, Mengelle C, Fourcade C, Marchou B, et al. Zika virus: high infectious viral load in semen, a new sexually transmitted pathogen. Lancet Infect Dis 2016; 16: 405.
13. Trioplex real-time RT-PCR assay. CDC 2017. https://www.fda.gov/downloads/medicaldevices/safety/emergencysituations/ucm491592.pdf.
14. Song J, Mauk MG, Hackett BA, Cherry S, Bau HH, Liu C. Instrument-free point-of-care molecular detection of Zika virus. Anal Chem 2016; 88: 7289–7294.
15. Song J, Liu C, Mauk MG, Rankin SC, Lok JB, et al. Two-stage isothermal enzymatic amplification for concurrent multiplex molecular detection. Clin Chem 2017; 63(3): 714–722.
The authors
Michael G Mauk PhD, Jinzhao Song PhD, Haim H. Bau PhD, Changchun Liu* PhD
Department of Mechanical Engineering and Applied Mechanics, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
*Corresponding author
E-mail: lchangc@seas.upenn.edu
Complete laboratory diagnosis of Zika virus infections
, /in Featured Articles /by 3wmediaLaboratory diagnosis of Zika virus (ZIKV) infections is based on two main pillars: direct detection of the viral RNA genome and serological detection of anti-ZIKV antibodies. Direct detection of the virus by reverse transcriptase real-time polymerase chain reaction (RT real-time PCR) is the most important method for diagnosing early acute infections. A new RT real-time PCR system with fully automated data evaluation provides highly standardized and streamlined detection of ZIKV RNA. Serology is useful for acute diagnostics as well as for longer term monitoring and epidemiological studies. An ELISA based on ZIKV NS1 antigen provides exceptionally high specificity with virtually no cross reactivity to other flaviviruses.
by Dr Jacqueline Gosink
Introduction
ZIKV has become firmly established in South and Central America and the Caribbean and is increasingly spreading to other parts of the world. The infection is now classified by the World Health Organization as an enduring public health challenge. Nearly one million people in 48 countries have been infected with ZIKV since the beginning of 2015, according to the Panamerican Health Organization. The actual number of cases is presumably much higher, since many infections are mild and go unreported. The virus is transmitted predominantly by mosquitos of the Aedes genus, which are ubiquitous in many topical and non-tropical regions. Transmission by sexual contact is also increasingly described. ZIKV infections are difficult to distinguish clinically from dengue virus (DENV) and chikungunya virus (CHIKV) infections, which manifest with similar symptoms of fever, exanthema and arthritis and are endemic in much the same geographic regions. There is, however, a growing body of evidence linking ZIKV to birth defects in fetuses and newborns and neurological complications such as Guillain-Barré syndrome in adults. Therefore, accurate diagnosis of ZIKV infections and differentiation between acute and past infections is critical for effective patient care.
ZIKV direct detection
The ZIKV RNA genome can be detected during the viremic phase of infection. The viral RNA is detectable for up to around 5 days after the onset of symptoms in serum and up to 10 days in urine. Molecular diagnostic detection is therefore highly effective for early diagnosis of ZIKV infections and discrimination of ZIKV from clinically similar infections such as DENV or CHIKV.
Novel RT real-time PCR assay
A new assay provides fast detection of ZIKV RNA in serum or urine by reverse transcriptase real-time polymerase chain reaction (RT real-time PCR) with fully automated data analysis. The EURORealTime Zika virus test is based on a one-tube reaction, comprising reverse transcription of the viral RNA into complementary DNA (cDNA) followed by PCR amplification and fluorescence-based real-time detection of defined sections of the ZIKV genome. The reverse transcription, amplification and detection of ZIKV cDNA are carried out by means of ZIKV-specific DNA primers and real-time DNA probes. RNA-based internal and positive controls verify the correct performance, integrity and functionality of the complete procedure. Ready-to-use reagents provide added reliability and convenience.
The evaluation of results is fully automated using the EURORealTime Analysis software and is therefore highly standardized and objective. All results, including those of the controls, are documented and archived. The software also supports simple and error-free test performance by guiding every step of the workflow. The entire detection procedure (excluding RNA extraction) takes less than 90 min.
Specifications and evaluation of the EURORealTime Zika virus test
Highest test sensitivity and specificity is ensured by the meticulous design of the primers and probes. Moreover, cross reactivity with other pathogens that may be present in serum or urine samples and/or are closely related to ZIKV has been excluded experimentally.
In clinical evaluation, 29 serum and 26 urine samples from patients with suspected ZIKV infection were analysed using the EURORealTime Zika virus and another CE/IVD-labelled ZIKV test system. There was a positive agreement of 95.2% and a negative agreement of 97.0% between the results obtained with the two tests (Table 1).
ZIKV serology
Serological detection is effective from soon after symptom onset (4-7 days) to beyond convalescence. Serology serves as a supplement to RT-PCR in acute cases. It is especially useful in cases where viral RNA is no longer detectable, for example if the infection is resolved or has moved into the chronic phase. Serological detection is particularly relevant in prenatal diagnostics, sexual healthcare and epidemiological surveys. Pregnant women with serological evidence of an infection can be offered intense prenatal monitoring, while seronegative women may be spared unnecessary worry. Due to the lengthy presence of ZIKV in semen, men who have resided in or travelled in endemic regions are advised to abstain from unprotected sexual intercourse for six months after returning to prevent sexual transmission, especially when their partner is or could be pregnant. Serological testing can be helpful in these cases for excluding or identifying an infection. As ZIKV continues to move into previously unaffected areas, epidemiological studies using serological methods can help to monitor the spread of the virus and probe its associated complications.
Relevance of immunoglobulin classes
Primary acute ZIKV infections are generally characterized by the occurrence of specific IgM antibodies, with IgG appearing at the same time or shortly afterwards. IgM can remain detectable for several months, while IgG is assumed to persist lifelong. Detection of specific IgM or a rise in the specific IgG titre in a pair of samples taken at least 7 to 10 days apart is evidence of an acute infection.
In secondary flavivirus infections, for example following a previous vaccination or infection with another flavivirus, specific IgM is often found at a low or undetectable titre. Therefore, additional tests like the detection of IgG or plaque reduction neutralization test are recommended.
Specific IgA may also be useful for diagnostics. In secondary flavivirus infections synthesis of IgG is rapidly stimulated. Shortly after infection the IgG titre levels off and is indistinguishable from IgG titres in convalescent infections, making seroconversion difficult to detect. This pattern has been observed in ZIKV patients from regions endemic for other flaviviruses. IgA has recently been proposed as a putative additional marker of acute infection in cases where IgM is not detectable and the IgG titre is already high.
Highly specific NS1-based ZIKV ELISA
Serological diagnosis of ZIKV is challenging due to the high cross-reactivity between flavivirus antibodies. This obstacle has been overcome by the use of recombinant non-structural protein 1 (NS1) from ZIKV as the antigenic substrate in ELISA. Use of this antigen avoids the cross-reactivity typically associated with tests based on whole virus antigens or viral glycoproteins. The NS1-based ELISA provides highly sensitive and specific ZIKV diagnostics, as demonstrated in numerous studies.
Clinical evaluation of IgM/IgG ELISA
The NS1-based Anti-Zika Virus ELISA was used to examine anti-ZIKV antibodies of classes IgG and IgM in various serum panels. In samples from patients with RT-PCR-confirmed infections (n=71), taken 5 days or more after symptom onset, the sensitivity of the test amounted to 100% for IgG/IgM (Table 2) (1). In a panel of blood donors the specificity of the ELISA was 99.8%.
In studies with a total of over 450 patients harbouring other arboviral infections, including DENV, CHIKV, tick-borne encephalitis virus (TBEV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and individuals vaccinated against yellow fever virus (YFV) or TBEV, the specificity lay between 96% and 100% (Table 3) (1, 2). In particular, a specificity of 100% was observed in DENV- and CHIKV-infected patients, demonstrating the suitability of the ELISA for discriminating these infections. In a further study (3) the Anti-Zika Virus ELISA showed no cross reactivity (100% specificity) in sera from patients with early convalescent DENV infections or suspected secondary DENV infections.
Usefulness of IgA testing
In a recent study investigating the diagnostic usefulness of IgA antibodies, anti-ZIKV antibodies of class IgA, IgM and IgG were analysed at serial time points in patients with confirmed ZIKV infections (4, 5). In two German travellers, IgM was detected early in infection as expected, followed by IgG seroconversion. IgA antibodies showed an initial increase and subsequent decrease. In two Columbian patients with a presumptive background of past flavivirus infection, IgM was persistently below the cut-off in both NS1-based and full virus-based tests, while IgG was already positive within the first week. Analysis of IgA in these patients demonstrated a titre increase, which peaked above the cut-off in week three and four before dropping below the threshold again (Figure 1). Thus, specific IgA may be useful for the diagnosis of acute infections and discrimination from past infections in IgM-negative patients.
Clinical evaluation of IgA ELISA
The NS-1-based Anti-Zika Virus ELISA was used to analyse anti-ZIKV antibodies of class IgA in Columbian patients (n=31) seven to ten days after positive ZIKV RT-PCR. 29 of the patients were positive for anti-ZIKV IgA, representing a sensitivity of 94%. The specificity of the IgA ELISA amounted to 97% in a control panel of German travellers with confirmed DENV infections and 100% in healthy blood donors and patients with other diseases. With the IgA ELISA, as with the IgM and IgG ELISAs, cross reactivity with antibodies against other flaviviruses, including DENV, TBEV, JEV, WNV and YFV, is almost completely avoided.
Differential diagnostics by IIFT
The indirect immunofluorescence test (IIFT) based on virus-infected cells offers an alternative sensitive screening assay for ZIKV antibodies. Automated microscopy and evaluation of results using the EUROPattern system streamlines the procedure. The ZIKV substrate can be combined with other substrates as a BIOCHIP mosaic, enabling potential cross-reactive antibodies or relevant differential diagnostic parameters to be investigated in parallel. In addition to ZIKV, available substrates include DENV (serotypes 1, 2, 3 and 4) and other flaviviruses (e.g. TBEV, YFV and JEV), as well as other arboviruses (e.g. CHIKV). Endpoint titration of the patient serum provides an indication of the virus causing the infection. As cross reactivity is common in patients with secondary flavivirus infections, BIOCHIP flavivirus mosaics are most useful for patients in non-epidemic countries, for example travellers returning from epidemic regions.
Perspectives
The swift development of sensitive and specific tests for ZIKV antibodies and ZIKV RNA has facilitated the diagnosis and surveillance of this rapidly emerging disease. The EUROIMMUN Anti-Zika Virus ELISA based on NS1 antigen is currently the only commercial serological test whose extremely high specificity has been described in various publications. It is, moreover, the first commercial serological ZIKV test to receive CE Mark (Europe; IgA, IgM and IgG) and ANVISA (Brazil; IgM, IgG, soon also IgA) registrations. The assay is fully automatable, making it ideal for high-throughput application in a routine setting. For direct detection of viral RNA, the new EURORealTime Zika virus test provides software-supported test performance and fully automated result evaluation and documentation, in contrast to many manual ZIKV RT-PCR tests. As ZIKV will likely remain a global health challenge in the foreseeable future, state-of-the-art test systems like these are crucial for monitoring the spread, improving diagnosis and elucidating the mechanisms of this challenging emerging disease.
References
1. Steinhagen et al. Euro Surveill. 2016 15;21(50). pii: 30426.
2. Huzly et al. Euro Surveill 2016;21(16):pii=30203.
3. Granger et al. Poster at the 32nd Clinical Virology Symposium (Florida, USA) 2016
4. Steinhagen et al. Poster at the IMED International Meeting on Emerging Infectious Diseases and Surveillance (Vienna, Austria) 2016
5. Steinhagen et al. Poster at the 1st International Conference on Zika Virus (Washington DC, USA) 2017
The author
Jacqueline Gosink, PhD
EUROIMMUN AG, Seekamp 31,
23560 Luebeck, Germany
www.euroimmun.com
Quality control testing on a random access molecular diagnostics platform running quantitative viral load assays
, /in Featured Articles /by 3wmediaThe DxN VERIS Molecular Diagnostics System* from Beckman Coulter is a real-time PCR analyser for accurate and precise quantitative detection of both RNA and DNA targets. Single sample random access offers workflow flexibility and automation benefits to the laboratory. The design features of the DxN VERIS System and performance characteristics of the VERIS HCV, HIV-1, HBV, and CMV viral load assays enable laboratories to develop Quality Control (QC) programmes tailored to their unique needs. Methods: A QC programme was developed by the Virology lab at the Rennes University Hospital, France. The laboratory evaluated the performance levels of the DxN VERIS System as well as the total number of VERIS HIV-1, HBV, and CMV tests performed over a period of five months. Results: The precision observed over the five-month study period was less than 5.8% CV with standard deviation (SD) within 0.16 log IU/ml. Based on these results the laboratory concluded that performing three levels of QC (negative, low, high) two times per week would provide an acceptable level of system control while significantly reducing QC costs and hands-on time.
Introduction
Consistency in reporting quantitative viral load results is critically important to clinical laboratories, physicians, and the patients they serve. The use of quantitative tests to measure viral load levels in patient samples is especially important for monitoring treatment. With the advent of new quantitative PCR (qPCR) assays for viral load testing, physicians are better able to manage diseases with antiretroviral therapy (ART).
Clinical laboratories are challenged to achieve stringent Quality Control (QC) objectives for viral load testing in an effective and economical manner. The use of external quality controls (EQC) provides laboratories with a means of monitoring variation in the analytical process as well as environmental factors that can affect patient results. In addition, EQC can assist laboratories in identifying when errors are occurring that can impact the utility of viral load assays. For these reasons, manufacturers of qPCR systems may recommend the use of EQC as part of the analytical process for viral load testing.
The DxN VERIS System and VERIS viral load assays are designed to deliver a high standard of clinical performance while providing rapid, convenient, and cost effective QC alternatives to the laboratory.
Quality control for quantitative diagnostic systems – a statistical approach
Statistical QC is defined as a procedure in which stable samples are measured and the observed results compared with limits that describe the variation expected when the measurement method is working properly[2]. Statistical QC is important to ensure the quality of the test results produced by any measurement method. An important concept in statistical QC is the definition of an “analytical run”. With many modern analytical systems, the definition of a run is not always clear. For example, many molecular diagnostics analysers available to laboratories today perform testing in “batch” mode, wherein each run corresponds to a single batch of several tests. While these methods can provide efficiencies in some testing environments (e.g. high volume labs) they can result in delayed results while the laboratory waits to accrue sufficient samples to complete the batch. In addition, batch systems lack the flexibility to adapt to fluctuating testing demand driven by sample volume and clinical needs in the laboratory. New qPCR systems are now available that provide “random access” capability; enabling labs to test individual samples at the precise time that they are most needed. In addition to providing more timely results for physicians and patients, these systems can also increase laboratory work flow efficiency, resulting in less hands-on time. For random access systems, an analytical run can be better understood in terms of the time or number of measurements for which the measurement is stable[2]. Statistical guidance for molecular assays typically suggests that quality control samples should be run at least once during each user-defined analytical run.
The DxN VERIS system
The DxN VERIS System is a fully automated molecular diagnostic system that integrates nucleic acid extraction, reaction setup, real-time PCR amplification and detection, and results interpretation into one system; saving space and time. The system provides single sample random access capability which allows the laboratory to run the right viral load test at the right time for physicians and patients. The DxN VERIS System provides time and workflow advantages compared to batch systems which require the laboratory to accrue a number of patient samples prior to each run.
Designed for quality and accuracy
The DxN VERIS System is engineered to deliver a high level of reliability and process control. The system provides a comprehensive range of individual process checks throughout the analytical process, from sample introduction to result reporting. Listed below are key features of the DxN VERIS System that ensure consistent performance and process control. Collectively, these capabilities may serve to reduce risk of analytical error within run and between runs.
Sample introduction
Nucleic acid extraction
Real-time PCR amplification and detection
An internal process control (PC) is run with each sample to monitor the reaction. The PC may be a plasmid or an inactivated virus that contains a selected or engineered target sequence and is designed to mimic the behaviour of the assay target throughout the extraction, purification, and PCR process.
In addition to these quality features, the DxN VERIS System displays QC results in chart format to provide a graphical view of the data. Depending on the characteristics of the data, the system uses a Levy-Jennings chart or a Shewhart chart. Multiple data sets can be viewed simultaneously in an overlay chart, or in up to four individual charts. On- board QC management software flags when QC is out of range.
QC procedure for VERIS viral load assays
Beckman-Coulter’s QC procedure provides a method of monitoring system performance while minimizing hands-on time and QC cost to the laboratory. Beckman Coulter recommends that Quality Control should be run in each 24-hour period in which test samples are run until variability limits have been established on the DxN VERIS System. Reduced frequency of control testing should be based on data as determined by the individual laboratory. Quality control materials should incorporate the analyte and a negative control.
Each laboratory should establish mean values and acceptable ranges to assure proper performance. Quality control results that do not fall within acceptable ranges may indicate invalid test results. It is recommended that laboratories examine all test results generated after obtaining the last acceptable quality control test point for this analyte.
In some countries or geographic locations, government regulation may define specific requirements that dictate frequency and number of QC data points and specimens used. Each laboratory should establish its own QC protocol based on data as determined by the laboratory in accordance with accrediting organizations and government regulations, as applicable[2].
Customer case study – Rennes University Hospital
Quality Control programmes utilizing the DxN VERIS System have been successfully implemented at customer laboratories across Europe. Described below is an example of a QC protocol developed in the Virology laboratory at Rennes University Hospital, France.
Rennes University Hospital is a 2,000 bed facility serving the Brittany region of France. The Virology lab processes approximately 133,000 analyses per year including 8,000 qPCR tests for HIV-1, HBV, and CMV viral load monitoring. The laboratory adopted the DxN VERIS system in 2016 based on the system’s workflow advantages and assay performance.
The analytical performance of the DxN VERIS System enabled the laboratory to consider the possibility of reducing the frequency of QC testing required to monitor routine patient analyses. In order to determine an appropriate QC frequency for viral load testing, the laboratory evaluated the performance characteristics of the analyser as well as the number of tests performed over a period time. Based on this assessment the laboratory concluded that performing three levels of QC (negative, low, high) two times per week would provide an acceptable level of system control while significantly reducing QC costs and hands-on time. This level of QC testing was appropriate based on the volume of tests performed by the laboratory. For labs that perform a higher volume of tests, QC may need to be performed more frequently in order to provide a sufficient level of QC relative to the number of tests performed. In case of QC out-of range, a procedure has been set-up in Rennes in order to re-test all samples analysed between the two QC measurement times. To validate the twice-weekly QC protocol, the lab evaluated the precision performance of each assay over 5 months. The precision observed over this time frame was less than 5.8% CV with standard deviation (SD) within 0.16 log IU/ml. No values were observed outside of the expected range. These data, summarized below, were determined by Rennes to be sufficient to support the twice-weekly QC protocol.
The Virology laboratory at Rennes University Hospital has been following the twice-weekly QC protocol since April 2016. This has resulted in a reduction in the cost per reportable result and simplified the QC process without impacting the quality of results produced by the lab.
Conclusion
Beckman Coulter’s DxN VERIS System provides a high level of assay performance, ease of use, and workflow efficiency. Effective quality control programs can be developed based on the unique testing requirements of each laboratory, resulting in a high level of system control while reducing hands-on time and QC cost.
* DxN VERIS products are CE-marked IVDs. DxN VERIS product line has not been submitted to U.S. FDA and is not available in the U.S. market. DxN VERIS Molecular Diagnostics System is also known as VERIS MDx Molecular Diagnostics System and VERIS MDx System.
The authors
www.beckmancoulter.comJ. Wyatt, P. Le Roux, V. Thibault1
Beckman Coulter Diagnostics | Brea, CA
1 Rennes University Hospital, France
2 CLSI. Statistical Quality Control for Quantitative Measurement Procedures:
Principles and Definitions; Approved
Guideline-Third Edition. CLSI document C24-A3. Wayne, PA: Clinical and
Laboratory Standards Institute.
DT 100 – The Dual Technology System
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